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Varroa destructor is widely recognized as a significant contributor to colony collapse disorder. Chemical acaricides, such as amitraz, have been extensively used for Varroa control due to their selectivity within beehives. However, the increasing number of cases of amitraz resistance across global V. destructor populations poses a significant challenge. In this study, we conducted a comprehensive molecular screening of the β-adrenergic-like octopamine receptor (Octβ2R), the target-site of amitraz, across 66 Turkish and 63 Belgian V. destructor populations. Although previously reported amitraz resistance mutations were not detected, the screening revealed a novel Y337F mutation located within transmembrane 7 (TM7) of Octβ2R in Turkish Varroa populations. Notably, this mutation was identified in the last residue of the highly conserved NPxxY motif associated with the activation of G-protein coupled receptors (GPCR). Among the 66 Varroa samples from Türkiye, twenty harbored the Y337F mutation, with eight samples exhibiting fixation of the mutation. Subsequent bioassays revealed over 8-fold resistance to amitraz in populations that contain the Y337F mutation. Genotyping of mites after exposure to 10 mg a.i./l amitraz demonstrated that all surviving mites were homozygous for the Y337F mutation, whereas dead mites carried susceptible alleles, providing genetic linkage between mutation and phenotype. Further, we used CRISPR-Cas9 editing to introduce the Y337F mutation in the orthologous Octβ2R of the model organism Tetranychus urticae. Crispants exhibited over threefold resistance to amitraz. In conclusion, this study identified and validated a novel amitraz resistance mutation. Additional research is required to further evaluate the phenotypic strength of Y337F in the context of operational resistance with current treatment strategies., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The spider mite Tetranychus urticae is a major agricultural pest with a global distribution, extremely diverse host range and a remarkable ability to develop resistance to a wide variety of acaricides. P450 mono-oxygenases have been frequently associated with resistance development in this species. In particular enzymes of the CYP392A-subfamily were shown to metabolize a number of key acaricides, including abamectin, amitraz, fenpyroximate and the active metabolite of pyflubumide. However, transcriptomic studies comparing highly resistant and susceptible populations have often revealed high expression of members of the CYP392D-subfamily, but these have been only poorly studied. Here, we conducted a meta-analysis of gene expression data of 20 populations and identified two key enzymes of this family, CYP392D2 and CYP392D8, whose expression is associated with resistance. We subsequently functionally expressed these enzymes, together with CYP392A11 and CYP392A16 as known metabolizers, and compared their potential to accept a wide diversity of acaricides as substrate. This study overall confirms previous discovered substrates for CYP392A11 and CYP392A16, but also reveals unreported metabolic activity towards new acaricides. These include carbaryl, chlorpyrifos and etoxazole for CYP392A16 and carbaryl, chlorpyrifos and NNI-0711-NH pyflubumide for CYP392A11. For the newly studied CYP392D-family, we show that CYP392D2 metabolizes pyridaben, fenpyroximate, etoxazole and chlorpyrifos, while CYP392D8 metabolizes carbaryl, fenazaquin and tebufenpyrad. Last, we observed that both CYP392A- and CYP392D-subfamily enzymes activate chlorpyrifos to its corresponding oxon. Our study indicates that there is both overlap and specificity in the activity of A- and D-subfamily enzymes against acaricides and model substrates. With the recent advent of highly efficient CRISPR/Cas9 gene editing protocols in T. urticae, the way is now paved to conduct further genetic experiments revealing and quantifying the role of these enzymes in the resistance phenotype in field populations., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Eye development and function rely on precise establishment, regression and maintenance of its many sub-vasculatures. These crucial vascular properties have been extensively investigated in eye development and disease utilizing genetic and experimental mouse models. However, due to technical limitations, individual studies have often restricted their focus to one specific sub-vasculature. Here, we apply a workflow that allows for visualization of complete vasculatures of mouse eyes of various developmental stages. Through tissue depigmentation, immunostaining, clearing and light-sheet fluorescence microscopy (LSFM) entire vasculatures of the retina, vitreous (hyaloids) and uvea were simultaneously imaged at high resolution. In silico dissection provided detailed information on their 3D architecture and interconnections. By this method we describe successive remodeling of the postnatal iris vasculature, involving sprouting and pruning, following its disconnection from the embryonic feeding hyaloid vasculature. In addition, we demonstrate examples of conventional and LSFM-mediated analysis of choroidal neovascularization after laser-induced wounding, showing added value of the presented workflow in analysis of modelled eye disease. These advancements in visualization and analysis of the respective eye vasculatures in development and complex eye disease open for novel observations of their functional interplay at a whole-organ level., Competing Interests: Declaration of competing interest Authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The two-spotted spider mite Tetranychus urticae is a polyphagous pest with an extraordinary ability to develop acaricide resistance. Here, we characterize the resistance mechanisms in a T. urticae population (VR-BE) collected from a Belgian tomato greenhouse, where the grower was unsuccessful in chemically controlling the mite population resulting in crop loss. Upon arrival in the laboratory, the VR-BE population was established both on bean and tomato plants as hosts. Toxicity bioassays on both populations confirmed that the population was highly multi-resistant, recording resistance to 12 out of 13 compounds tested from various mode of action groups. DNA sequencing revealed the presence of multiple target-site resistance mutations, but these could not explain resistance to all compounds. In addition, striking differences in toxicity for six acaricides were observed between the populations on bean and tomato. The highest difference was recorded for the complex II inhibitors cyenopyrafen and cyflumetofen, which were 4.4 and 3.3-fold less toxic for VR-BE mites on tomato versus bean. PBO synergism bioassays suggested increased P450 based detoxification contribute to the host-dependent toxicity. Given the involvement of increased detoxification, we subsequently determined genome-wide gene expression levels of VR-BE on both hosts, in comparison to a reference susceptible population, revealing overexpression of a large set of detoxification genes in VR-BE on both hosts compared to the reference. In addition, a number of mainly detoxification genes with higher expression in VR-BE on tomato compared to bean was identified, including several cytochrome P450s. Together, our work suggests that multi-resistant field populations can accumulate a striking number of target-site resistance mutations. We also show that the host plant can have a profound effect on the P450-associated resistance levels to cyenopyrafen and cyflumetofen., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The development of insecticide resistance in malaria vectors is a challenge for the global effort to control and eradicate malaria. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the detoxification of many classes of insecticides. For mosquitoes, it is known that overexpression of an epsilon GST, GSTe2, confers resistance towards DDT and pyrethroids. In addition to GSTe2, consistent overexpression of a delta class GST, GSTd3, has been observed in insecticide resistant populations of different malaria vector species. However, the functional role of GSTd3 towards DDT resistance has not yet been investigated. Here, we recombinantly expressed both GSTe2 and GSTd3 from Anopheles arabiensis and compared their metabolic activities against DDT. Both AaGSTd3 and AaGSTe2 exhibited CDNB-conjugating and glutathione peroxidase activity and DDT metabolism was observed for both GSTs. However, the DDT dehydrochlorinase activity exhibited by AaGSTe2 was much higher than for AaGSTd3, and AaGSTe2 was also able to eliminate DDE although the metabolite could not be identified. Molecular modeling revealed subtle differences in the binding pocket of both enzymes and a better fit of DDT within the H-site of AaGSTe2. The overexpression but much lower DDT metabolic activity of AaGSTd3, might suggest that AaGSTd3 sequesters DDT. These findings highlight the complexity of insecticide resistance in the major malaria vectors and the difficulties associated with control of the vectors using DDT, which is still used for indoor residual spraying., Competing Interests: Declaration of Competing Interest The authors declare no competing of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The stimulation of biological processes by sublethal doses of insecticides or other stressors is known as hormesis. Here, we have evaluated whether exposure to field-relevant or low concentrations of neonicotinoids induce changes in the reproductive capacity of the bulb mite Rhizoglyphus robini (Acari: Acaridae). Among the tested neonicotinoids imidacloprid, thiamethoxam, and dinotefuran, the highest hormetic effect on the reproduction of R. robini occurred 24 h after the 48 h exposure period to imidacloprid at concentrations of 70 and 140 mg a.i./L. Despite the stimulating effects of imidacloprid on mite reproduction, no significant differences were observed in the offspring (F 1 ) for biological aspects including egg hatch rate, embryonic period and sex ratio, while an increase was found in the duration of development time from egg to adult. Evaluation of the detoxification enzyme activities of treated adults showed that the highest activity of carboxyl/cholinesterases, cytochrome P450s, and glutathione S-transferases was obtained when exposed to 70, 140 and 70 mg a.i./L imidacloprid, immediately after the exposure period, respectively. Also, an increase in the activity of the antioxidant enzyme catalase was observed compared to that of the control. After imidacloprid pretreatment (140 mg a.i./L), the tolerance of adult mites to diazinon was increased about two-fold. This study shows that exposure to imidacloprid can induce hormetic effects on R. robini and could severely complicate its control due to a higher reproduction, enhanced detoxification enzyme activities, and increased tolerance against other pesticides., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Acequinocyl and bifenazate are potent acaricides acting at the Q o site of complex III of the electron transport chain, but frequent applications of these acaricides have led to the development of resistance in spider mites. Target-site resistance caused by mutations in the conserved cd1- and ef-helices of the Q o pocket of cytochrome b has been elucidated as the main resistance mechanism. We therefore monitored Q o pocket mutations in European field populations of Tetranychus urticae and uncovered a new mutation, L258F. The role of this mutation was validated by revealing patterns of maternal inheritance and by the independently replicated introgression in an unrelated susceptible genetic background. However, the parental strain exhibited higher resistance levels than conferred by the mutation alone in isogenic lines, especially for acequinocyl, implying the involvement of strong additional resistance mechanisms. This was confirmed by revealing a polygenic inheritance pattern with classical genetic crosses and via synergism experiments. Therefore, a genome-wide expression analysis was conducted that identified a number of highly overexpressed detoxification genes, including many P450s. Functional expression revealed that the P450 CYP392A11 can metabolize bifenazate by hydroxylation of the ring structure. In conclusion, the novel cytochrome b target-site mutation L258F was uncovered in a recently collected field strain and its role in acequinocyl and bifenazate resistance was validated. However, the high level of resistance in this strain is most likely caused by a combination of target-site resistance and P450-based increased detoxification, potentially acting in synergism., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is the most economically important mite pest in agricultural areas and chemical acaricides are widely used to control T. urticae populations. Cyflumetofen is a recently introduced acaricide that inhibits the mitochondrial electron transport chain at complex II (succinate dehydrogenase, SDH), which represents the most recently developed mode of action for mite control worldwide. In the present study, started upon the launch of cyflumetofen in Turkey, a five-year survey was performed to monitor cyflumetofen susceptibility in 28 T. urticae populations collected from agricultural fields across the country. The first resistance case that might cause control failure in practical field conditions was uncovered in 2019, three years after the registration of cyflumetofen. In addition, an extremely resistant population (1722-fold resistance) was also detected towards the end of 2019. Cyflumetofen resistance did not decrease in the laboratory after relaxation of selection pressure for over one year in field-collected populations, suggesting the absence of a fitness cost associated with resistance in these populations. Next to phenotypic resistance, metabolic and physiological mechanisms underlying the decreased susceptibility were also investigated. Synergism assays showed the involvement of P450 monooxygenases in cyflumetofen resistance. Downregulation of carboxylesterases as resistance mechanism, is underpinned by the fact that pre-treatment with esterase inhibitor DEF decreased cyflumetofen toxicity in field-collected strains. Furthermore, a novel H258L substitution in the subunit B of complex II was uncovered in a field population. In silico modeling of the new mutation suggested that the mutation might indeed influence toxicity to complex II inhibitors cyenopyrafen and pyflubumide, but most likely not cyflumetofen. However, further studies are needed to uncover the exact role of this mutation in resistance to this new class of complex II inhibitors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Essential oils (EOs) can provide important alternatives to chemical insecticides in the control of pests. In this study, 12 EOs of native plant species from Iran were evaluated for their adulticidal activity against the house fly. In addition, we examined the insecticidal activity of Zataria multiflora and Rosmarinus officinalis EOs on adult female house flies from pyrethroid and organophosphate resistant and susceptible populations, using both fumigant and topical bioassays. The involvement of detoxification enzymes in susceptibility was investigated with synergism experiments in vivo, while the inhibitory effects of R. officinalis and Zataria multiflora EOs on the activities of cytochrome P450-dependent monooxygenases (P450s), carboxylesterases (CarEs) and glutathione S-transferases (GSTs) were determined by enzymatic inhibition assays in vitro. The EOs of Z. multiflora, Mentha pulegium, R. officinalis and Thymus vulgaris were the most effective against adults in contact topical assays, while oils extracted from Eucalyptus cinerea, Z. multiflora, Citrus sinensis, R. officinalis, Pinus eldarica and Lavandula angustifolia where the most effective in fumigant assays. Rosmarinus officinalis and Z. multiflora EOs were selected for further investigation and showed higher toxicity against a susceptible population, compared to two insecticide-resistant populations. Correlation analysis suggested cross-resistance between these EOs and pyrethroids in the resistant populations. The toxicity of both EOs on the resistant populations was synergized by three detoxification enzyme inhibitors. Further, in vitro inhibition studies showed that R. officinalis and Z. multiflora EOs more effectively inhibited the activities of the detoxification enzymes from flies of the susceptible population compared to those of the pyrethroid resistant populations. Synergistic and enzymatic assays further revealed that increased activities of P450s, GSTs, and CarEs are possibly involved in the cross-resistance between EOs and pyrethroids. Investigating the molecular mechanisms of P450s, GSTs, and CarEs in the resistance to EOs should be subject to further studies., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Pyridaben is a mitochondrial electron transport complex I inhibitor. The H110R mutation in the PSST subunit has been reported as a major factor in pyridaben resistance in the two-spotted spider mite, Tetranychus urticae. However, backcross experiments revealed that the mutant PSST alone conferred only moderate resistance. In contrast, inhibition of cytochrome P450 (CYP) markedly reduces resistance levels in a number of highly resistant strains. It was reported previously that maternal factors contributed to the inheritance of pyridaben resistance in the egg stage, but the underlying mechanisms have yet to be elucidated. Here, we studied the combined effects of the PSST H110R mutation and candidate CYPs, as metabolic resistance factors, on pyridaben resistance in T. urticae. We found that the maternal effects of inheritance of resistance in the egg stage were associated with CYP activity. Analysis of differential gene expression by RNA-seq identified CYP392A3 as a candidate causal factor for the high resistance level. Congenic strains, where the alleles of both PSST and CYP392A3 were derived from a resistant strain (RR&#95;i; i = 1 or 2) and a susceptible strain (SS&#95;i) in a common susceptible genetic background, were constructed by marker-assisted backcrossing. RR&#95;i showed upregulation of CYP392A3 and high resistance levels (LC 50  > 10,000 mg L -1 ), while SS&#95;i had LC 50  < 10 mg L -1 . To disentangle the individual effects of PSST and CYP392A3 alleles, we also attempted to uncouple these genes in RR&#95;i. We conclude that given the variation in LC 50 values and expression levels of CYP392A3 in the congenic and uncoupled strains, it is likely that the high pyridaben resistance levels are due to a synergistic or cumulative effect of the combination of mutant PSST and associated CYPs, including CYP392A3, but other yet to be discovered factors cannot be excluded., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The predatory mite Neoseiulus californicus McGregor (Acari: Phytoseiidae) is an important natural enemy of phytophagous mites, and naturally established populations are often found in apple orchards. However, insecticide applications to control pests cause side effects to non-target organisms such as N. californicus. Pirimicarb, a widely used carbamate insecticide in apple orchards, is generally considered a selective aphidicide, however, toxicity to beneficial insects and predatory mites has been reported. Furthermore, the molecular basis for this selectivity, if present in N. californicus, is still largely unknown. In this study, 8 field-collected N. californicus populations were investigated and showed up to 27-fold resistance compared to a susceptible laboratory population. Selection in the laboratory for 5 consecutive generations resulted in a 69-fold pirimicarb resistance. Although there were no significant difference in terms of the acetlycholinesterase (AChE) activity between susceptible and field-collected populations, the selected population exhibited a significantly higher AChE activity. In addition, gene copy number variation of acetylcholinesterase (ace) gene among populations was detected and ranged from 1.6 to 2.1-fold relative to the susceptible population. All field-collected populations, but not the selected population, had a significantly higher ace copy number compared to the susceptible population (t-test, p < 0.05). Molecular analysis of the target-site (AChE) revealed, for the first time, a phenylalanine to tryptophan substition at position 331 in AChE (Torpedo californica numbering), both in field-collected and the selected population, but not in the susceptible population. Last, the selected F5 population consumed significantly more Tetranychusurticae adults than the parental population. Together, the results of this study shed light on the molecular determinants of acaricide selectivity in predatory mites, and will contribute to a better design of an integrated mite management program, including the use of pesticide resistant N. californicus in apple orchards., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The poultry red mite, Dermanyssus gallinae, is the most important blood sucking ectoparasite of egg laying hens and causes economic losses in poultry farms worldwide. Although various management methods exist, the control of poultry red mites (PRMs) mainly relies on acaricides such as pyrethroids and organophosphates (OPs) in many regions of the world. However, repeated use of these synthetic chemicals has resulted in resistance development causing control failure of PRM. In this study, we investigated acaricide resistance mechanisms of Turkish PRM populations. First, we obtained the COI sequence from 30 PRM populations from different regions in Turkey and identified four different COI haplotypes. Toxicity assays showed that four field-collected PRM populations were highly resistant to the pyrethroid alpha-cypermethrin, with resistance ratios (RRs) varying between 100- and 400-fold, while two of these populations had a RR of more than 24-fold against the OP acaricide phoxim. Biochemical assays showed a relatively higher activity of glutathione-S-transferases and carboxyl-cholinesterases, two well-known classes of detoxification enzymes, in one of these resistant populations. In addition, we also screened for mutations in the gene encoding the voltage-gated sodium channel (vgsc) and acetylcholinesterase 1 (ace-1), the target-site of pyrethroids and OPs, respectively. In all but two PRM populations, at least one vgsc mutation was detected. A total of four target-site mutations, previously associated with pyrethroid resistance, M918T, T929I, F1534L, F1538L were found in domain II and III of the VGSC. The T929I mutation was present in the vgsc of almost all PRM populations, while the other mutations were only found at low frequency. The G119S/A mutation in ace-1, previously associated with OP resistance, was found in PRM for the first time and present in fourteen populations. Last, both alive and dead PRMs were genotyped after pesticide exposure and supported the possible role of target-site mutations, T929I and G119S, in alpha-cypermethrin and phoxim resistance, respectively. To conclude, our study provides a current overview of resistance levels and resistance mutations in Turkish PRM populations and might aid in the design of an effective resistance management program of PRM in Turkey., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pesticide resistance in spider mites drives the development of acaricides with novel mode of action, which could benefit from RNAi as a screening tool in search of new molecular targets. RNAi via oral delivery of dsRNA has been frequently reported in spider mites, but injection of dsRNA is rarely reported. We compare here the efficiency of oral delivery versus injection of dsRNA in female adult mites. When comparing silencing efficiency, oral delivery of dsRNAs silenced 40.6 ± 8.9% of CPR, 63.8 ± 6.9% of CHMP2A, and 37.7 ± 5.7% of CHMP3 genes. Similar silencing efficiencies were found for injection (48.6 ± 3.7% of CPR, 70.2 ± 4.1% of CHMP2A, 59.8 ± 2.2% of CHMP3), but with much lower quantities of dsRNAs. Oral delivery of dsRNA failed to silence the expression of the CHMP4B gene, but this could be accomplished by injection of dsRNA (23.1 ± 1.0%). When scoring the phenotypic effects of silencing, both oral delivery and injection of CHMP2A- and CHMP3-dsRNA influenced the locomotion speed of mites significantly. For CPR, silencing could only be accomplished by dsRNA injection, not by feeding. CPR silencing significantly impacted the toxicity of a typical acaricide, pyridaben, as the susceptibility of mites raised 2.75-fold. Last, injection of Eya-dsRNA in adults produced transgenerational phenotypic effects on 3.59% of offspring, as quantified by an observed deviation in eye development, while oral delivery of Eya-dsRNA did not. In conclusion, injection of dsRNA is superior to oral delivery in silencing the expression of the selected genes in this study and could be considered the method of choice to study gene function in reverse genetic approaches., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The intense spraying of pesticides to control different arthropod pests has resulted in negative side effects for the management of pests. It was previously discovered that exposure to non-acaricidal insecticides alone or in a mixture, results in lower efficiency of the acaricide spirodiclofen used for Brevipalpus spp. control. We investigate here whether the induced expression of detoxification enzymes by non-lethal insecticides may antagonize spirodiclofen toxicity. Brevipalpus californicus mites exposed to the insecticide phosmet alone or in combination with spirodiclofen showed increased activity of P450 monooxygenases (P450s). No antagonistic effects in mite mortality were observed by the combination of phosmet and spirodiclofen. On the other hand, mites exposed to the insecticide imidacloprid alone or in combination with spirodiclofen showed an increase in the activity of P450s, carboxylcholinesterases (CCE), and glutathione-S-transferases (GST). An antagonistic effect on mite mortality was observed when mites were exposed to the LC 25 of spirodiclofen combined with the field rate treatment of imidacloprid. The addition of PBO (a P450 monooxygenase inhibitor) to the mixture of spirodiclofen and imidacloprid resulted in a synergistic effect over mite mortality but the addition of DEM (a GST inhibitor) resulted in an antagonist effect. Taken together, this study showed that the combination of imidacloprid with spirodiclofen is antagonistic for the control of B. californicus, and this results from the induction of detoxification enzymes, such as P450s, CCE, and GST. The use of inhibitors highlights the role of these enzymes in the antagonism of the mixture., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chemical insecticides are a major tool for the control of many of the world's most damaging arthropod pests. However, their intensive application is often associated with the emergence of resistance, sometimes with serious implications for sustainable pest control. To mitigate failure of insecticide-based control tools, the mechanisms by which insects have evolved resistance must be elucidated. This includes both identification and functional characterization of putative resistance genes and/or mutations. Research on this topic has been greatly facilitated by using powerful genetic model insects like Drosophila melanogaster, and more recently by advances in genome modification technology, notably CRISPR/Cas9. Here, we present the advances that have been made through the application of genome modification technology in insecticide resistance research. The majority of the work conducted in the field to date has made use of genetic tools and resources available in D. melanogaster. This has greatly enhanced our understanding of resistance mechanisms, especially those mediated by insensitivity of the pesticide target-site. We discuss this progress for a series of different insecticide targets, but also report a number of unsuccessful or inconclusive attempts that highlight some inherent limitations of using Drosophila to characterize resistance mechanisms identified in arthropod pests. We also discuss an experimental framework that may circumvent current limitations while retaining the genetic versatility and robustness that Drosophila has to offer. Finally, we describe examples of direct CRISPR/Cas9 use in non-model pest species, an approach that will likely find much wider application in the near future., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Insecticides of the tetronic/tetramic acid family (cyclic ketoenols) are widely used to control sucking pests such as whiteflies, aphids and mites. They act as inhibitors of acetyl-CoA carboxylase (ACC), a key enzyme for lipid biosynthesis across taxa. While it is well documented that plant ACCs targeted by herbicides have developed resistance associated with mutations at the carboxyltransferase (CT) domain, resistance to ketoenols in invertebrate pests has been previously associated either with metabolic resistance or with non-validated candidate mutations in different ACC domains. A recent study revealed high levels of spiromesifen and spirotetramat resistance in Spanish field populations of the whitefly Bemisia tabaci that was not thought to be associated with metabolic resistance. We confirm the presence of high resistance levels (up to >640-fold) against ketoenol insecticides in both Spanish and Australian B. tabaci strains of the MED and MEAM1 species, respectively. RNAseq analysis revealed the presence of an ACC variant bearing a mutation that results in an amino acid substitution, A2083V, in a highly conserved region of the CT domain. F1 progeny resulting from reciprocal crosses between susceptible and resistant lines are almost fully resistant, suggesting an autosomal dominant mode of inheritance. In order to functionally investigate the contribution of this mutation and other candidate mutations previously reported in resistance phenotypes, we used CRISPR/Cas9 to generate genome modified Drosophila lines. Toxicity bioassays using multiple transgenic fly lines confirmed that A2083V causes high levels of resistance to commercial ketoenols. We therefore developed a pyrosequencing-based diagnostic assay to map the spread of the resistance alleles in field-collected samples from Spain. Our screening confirmed the presence of target-site resistance in numerous field-populations collected in Sevilla, Murcia and Almeria. This emphasizes the importance of implementing appropriate resistance management strategies to prevent or slow the spread of resistance through global whitefly populations., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The citrus red mite, Panonychus citri, is a major pest on citrus all around the world. Mitochondrial Electron Transport Inhibitors of complex I (METI-I) acaricides such as fenpyroximate have been used extensively to control P. citri populations, which resulted in multiple reports of METI-I resistant populations in the field. In this study, biochemical and molecular mechanisms of fenpyroximate resistance were investigated in P. citri. Seven populations were collected from Northern provinces of Iran. Resistance ratios were determined and reached up to 75-fold in comparison to a fenpyroximate susceptible population. Cross-resistance to two additional METI-I acaricides, pyridaben and tebufenpyrad, was detected. PBO synergism experiments, in vivo enzyme assays and gene expression analysis suggest a minor involvement of cytochrome P450 monooxygenases in fenpyroximate resistance, which is in contrast with many reported cases for the closely related Tetranychus urticae. Next, we determined the frequency of a well-known mutation in the target-site of METI-Is, the PSST subunit, associated with METI-I resistance. Indeed, the H92R substitution was detected in a highly fenpyroximate resistant P. citri population. Additionally, a new amino acid substitution at a conserved site in the PSST subunit was detected, A94V, with higher allele frequencies in a moderately resistant population. Marker-assisted back-crossing in a susceptible background confirmed the potential involvement of the newly discovered A94V mutation in fenpyroximate resistance. However, introduction of the A94V mutation in the PSST homologue of D. melanogaster using CRISPR-Cas9 did not result in fenpyroximate resistant flies. In addition, differences in binding curves between METI-Is and complex I measured directly, in isolated transgenic and wildtype mitochondria preparations, could not be found., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The European red mite Panonychus ulmi (Koch) is a major pest of apple trees worldwide and causes significant damage to apple orchards in Iran. Pyrethroid insecticides/acaricides, such as fenpropathrin and fenvalerate, are widely used to control P. ulmi, but their long-term use may lead to low efficacy. Earlier studies investigating pyrethroid resistance in closely related mites such as Tetranychus urticae revealed that pyrethroid resistance was associated with point mutations in the voltage-gated sodium channel gene (vgsc). The aim of this study was to investigate the biochemical and molecular mechanisms of fenpropathrin and fenvalerate resistance in Iranian populations of P. ulmi. Pyrethroid toxicity bioassays were carried out on different P. ulmi field populations. Marand (resistance ratio, RR = 149), Maraqeh (RR = 90) and Mianeh2 (RR = 71) populations exhibited high levels of resistance to fenpropathrin, compared to a susceptible field population (Shahin Dej). Resistance was also observed for fenvalerate with resistance ratio's ranging from 2- to 20-fold. Synergism experiments and enzyme activity assays predicted a minor role for classical detoxification enzymes. In contrast, two amino acid substitutions in the VGSC, L1024V and F1538I, that were previously shown to confer pyrethroid resistance, were detected in all three resistant P. ulmi populations and point towards target-site insensitivity as the most likely resistance mechanism. Furthermore, sequencing after cloning of vgsc fragments from single haploid males revealed the presence of multiple copies of vgsc in a highly resistant strain. The link between resistance mutations and vgsc copy number variation should be the subject of future study, as this might be used to develop molecular markers for monitoring pyrethroid resistance of P. ulmi in the field., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Α reduction of pyrethroid efficacy has been recently recorded in Bactrocera oleae, the most destructive insect of olives. The resistance levels of field populations collected from Crete-Greece scaled up to 22-folds, compared to reference laboratory strains. Sequence analysis of the IIS4-IIS6 region of para sodium channel gene in a large number of resistant flies indicated that resistance may not be associated with target site mutations, in line with previous studies in other Tephritidae species. We analyzed the transcriptomic differences between two resistant populations versus an almost susceptible field population and two laboratory strains. A large number of genes was found to be significantly differentially transcribed across the pairwise comparisons. Interestingly, gene set analysis revealed that genes of the 'electron carrier activity' GO group were enriched in one specific comparison, which might suggest a P450-mediated resistance mechanism. The up-regulation of several transcripts encoding detoxification enzymes was qPCR validated, focusing on transcripts coding for P450s. Of note, the expression of contig00436 and contig02103, encoding CYP6 P450s, was significantly higher in all resistant populations, compared to susceptible ones. These results suggest that an increase in the amount of the CYP6 P450s might be an important mechanism of pyrethroid resistance in B. oleae., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Onion thrips, Thrips tabaci Lindeman, is the main pest of onion-growing fields in Isfahan and is mainly controlled by frequently spraying several insecticides. To investigate the resistance status and mechanisms, the susceptibility of ten field populations collected from Isfahan onion-growing regions were tested to several currently used pesticides. Resistance to the tested insecticides was observed in most populations when compared with the susceptible reference population. Enhanced detoxification, implicated by the use of inhibitors of major metabolic detoxification enzymes, was observed in the populations resistant to profenofos and chlorpyrifos. In the deltamethrin resistant populations, the amino acid substitution T929I was detected in the voltage gated sodium channel, which is known to confer pyrethroid resistance. These data are a first step towards more efficient resistance management tactics through early detection of resistant onion thrips in Iran., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The recent accumulation of molecular studies on mutations in insects, ticks and mites conferring resistance to insecticides, acaricides and biopesticides is reviewed. Resistance is traditionally classified by physiological and biochemical criteria, such as target-site insensitivity and metabolic resistance. However, mutations are discrete molecular changes that differ in their intrinsic frequency, effects on gene dosage and fitness consequences. These attributes in turn impact the population genetics of resistance and resistance management strategies, thus calling for a molecular genetic classification. Mutations in structural genes remain the most abundantly described, mostly in genes coding for target proteins. These provide the most compelling examples of parallel mutations in response to selection. Mutations causing upregulation and downregulation of genes, both in cis (in the gene itself) and in trans (in regulatory processes) remain difficult to characterize precisely. Gene duplications and gene disruption are increasingly reported. Gene disruption appears prevalent in the case of multiple, hetero-oligomeric or redundant targets., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Acaricides are one of the cornerstones of an efficient control program for phytophagous mites. An analysis of the global acaricide market reveals that spider mites such as Tetranychus urticae, Panonychus citri and Panonychus ulmi are by far the most economically important species, representing more than 80% of the market. Other relevant mite groups are false spider mites (mainly Brevipalpus), rust and gall mites and tarsonemid mites. Acaricides are most frequently used in vegetables and fruits (74% of the market), including grape vines and citrus. However, their use is increasing in major crops where spider mites are becoming more important, such as soybean, cotton and corn. As revealed by a detailed case study of the Japanese market, major shifts in acaricide use are partially driven by resistance development and the commercial availability of compounds with novel mode of action. The importance of the latter cannot be underestimated, although some compounds are successfully used for more than 30 years. A review of recent developments in mode of action research is presented, as such knowledge is important for devising resistance management programs. This includes spirocyclic keto-enols as inhibitors of acetyl-CoA carboxylase, the carbazate bifenazate as a mitochondrial complex III inhibitor, a novel class of complex II inhibitors, and the mite growth inhibitors hexythiazox, clofentezine and etoxazole that interact with chitin synthase I., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The two-spotted spider mite Tetranychus urticae is one of the most important agricultural pests world-wide. It is extremely polyphagous and develops resistance to acaricides. The overexpression of several glutathione S-transferases (GSTs) has been associated with insecticide resistance. Here, we functionally expressed and characterized three GSTs, two of the delta class (TuGSTd10, TuGSTd14) and one of the mu class (TuGSTm09), which had been previously associated with striking resistance phenotypes against abamectin and other acaricides/insecticides, by transcriptional studies. Functional analysis showed that all three GSTs were capable of catalyzing the conjugation of both 1-chloro-2,4 dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene(DCNB) to glutathione (GSH), as well as exhibiting GSH-dependent peroxidase activity toward Cumene hydroperoxide (CumOOH). The steady-state kinetics of the T. urticae GSTs for the GSH/CDNB conjugation reaction were determined and compared with other GSTs. The interaction of the three recombinant proteins with several acaricides and insecticides was also investigated. TuGSTd14 showed the highest affinity toward abamectin and a competitive type of inhibition, which suggests that the insecticide may bind to the H-site of the enzyme. The three-dimensional structure of the TuGSTd14 was predicted based on X-ray structures of delta class GSTs using molecular modeling. Structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of TuGSTd14., (Copyright © 2015 Elsevier Inc. All rights reserved.)